It is generally accepted that DNA is the crucial target in the cytotoxic effects of ionising radiation. There is considerable evidence to support the view that DNA double-stranded (ds) breaks are particularly important. The DNA damage results from both direct ionisation in the DNA molecule (direct effect) and by indirect effects mediated by the radiolysis products of water. Carbon-centred radicals on the deoxyribose moiety of DNA are thought to be important precursors of strand breaks. Ionising radiation also induces damage in DNA bases. If the level of cellular DNA damage is sufficient, the consequence of irradiation is cell kill, and thus ionising radiation is used as a mode of cancer therapy. For irradiated normal tissues, the cell killing can result in temporary or permanent impairment of tissue and organ function. The extent of such effects is dependant upon the radiation dose, and if sufficient, can be lethal to the organism. For humans and other animals, hematopoiesis is the most radiosensitive organ/function, followed by the gastrointestinal mucosa. Even if radiation induced DNA damage is sublethal, mutagenic lesions can have serious long term consequences, including carcinogenesis.
The medical strategies or countermeasures aimed at reducing the extent of radiation-induced effects are broadly described as radioprotectors (which to be effective, generally need to be administered prior to radiation exposure), mitigants/mitigators (which can be effective if administered after irradiation, but before the appearance of symptoms), and treatments which are generally administered after the appearance of symptoms. A sub-class of the prophylactic radioprotectors are drugs that reduce the extent of the initial radiation-induced DNA damage, and it is this sub-class that is the major focus of the present invention.
The commercial potential of radioprotectors resides primarily in two distinct arenas. One of these relates to the need to protect normal tissues in cancer radiotherapy patients, and the other concerns the need to assuage the consequences of unplanned irradiation associated with civil scenarios, such as radiation accidents and radiation terrorism, as well as irradiation in military contexts. This second scenario would also include planned exposure to ionising radiation in medical diagnostic procedures, in which administration of radioprotectors could ameliorate the health risks associated with low or modest radiation doses, without interfering with diagnostic imaging processes.
The treatment of tumours with ionising radiation (hereinafter referred to as “cancer radiotherapy”) is used extensively in cancer therapy. The goal of such treatment is the destruction of tumour cells and inhibition of tumour cell growth presumably through DNA damage, while minimising damage to non-tumour cells and tissues. The potential for damage to non-tumour cells in the vicinity of the tumour limits the radiation dose that can be administered, which in turn often limits the effectiveness of radiotherapy against certain tumours. This is especially the case in relation to brain tumours and tumours in the abdominal cavity.
Cancer radiotherapy is a very significant public health activity. Given the incidence of cancer in the population and the international assessment that more than 50% of cancer patients benefit from inclusion of radiotherapy in their treatment, more than 10% of the population are likely to experience cancer radiotherapy in their lifetime.
The dominant consideration in prescribing radiation doses for cancer radiotherapy is the assessment of tolerance of the most radiosensitive normal tissues/organs in the treatment field. This assessment, together with the expected radiation dose required to eradicate a tumour, often determines whether the treatment strategy is aimed at cure or palliation. In many cases, the maximum tolerable doses are insufficient to eradicate the tumour. This dilemma is embodied in the concept of therapeutic ratio, which represents the ratio of probabilities of tumour control versus normal tissue morbidity. Approaches to improving the therapeutic ratio include:                (a) optimising the physical targeting of the radiation to the tumour;        (b) fractionation of the radiation dose; and        (c) the use of radiomodifiers (which includes both radio-protectors and radiosensitisers, the latter of which can be used to increase the level of cell kill per unit of radiation dose).        
Improving the physical delivery of radiation has had a considerable impact on the practice of radiotherapy. For example, increasing the energy of x-ray photons from several hundred kilovolts to the present-day megavoltage beams enables the zone of maximum radiation dose to be set at depths of several centimetres, whereas with the older machines the maximum dose was near the skin surface. There are a number of more sophisticated approaches to “tailoring” treatment beams in various stages of development and implementation. Brachytherapy, the use of implanted radioactive sources rather than external beams, is a further approach to improving the physical dose distribution.
Almost without exception, curative external beam radiotherapy involves fractionation of the radiation dose. An example of a conventional schedule would be a total of 60 Gray given in thirty 2 Gray fractions. Since cells have the capacity to repair radiation damage between fractions, the fractionated treatment results is much less cell-kill than a single dose of 60 Gray. However, normal cells generally have a greater repair capacity than do tumour cells, so the “sparing” effect of fractionation is more marked for normal tissues. In short, fractionation improves the therapeutic ratio.
Exploration of radiomodifiers such as radioprotectors and radiosensitisers has focussed on hypoxic cell sensitisers such as metranidazole and misonidazole. Radioprotectors have received much less attention than radiosensitisers at the clinical level. The nuclear era spawned considerable effort in the development of radioprotectors with more than 4000 compounds being synthesised and tested at the Walter Reed Army Institute of Research in the United States of America in the 1960's. With the exception of a compound that was called WR2728 (later called Ethyol and now known as Amifostine) none of the compounds have proved useful for cancer radiotherapy, and even WR2728 was considered too toxic for administration in either the military or industrial contexts (i.e., protection against total body irradiation). More recently, for example, Metz and co-workers (Metz et al, Clin Cancer Res. 10, 6411-17, 2004) (15) developed the radioprotective compound known as TEMPOL, which demonstrates only limited efficacy even at very high concentrations, and Burdelya and colleagues (Burdelya et al Science 320, 226-30, 2008) (16) developed the compound known as the TOLL receptor agonist which suffers from the necessity for it to be administered systemically.
It is important to note the interplay between the three approaches (a)-(c), above, to improving the therapeutic ratio. A combination of improved physical targeting, fractionation and radiomodifiers could transform the intent in some radiotherapy situations from palliative to curative. For curative schedules, successful application of radiomodifiers would relax the requirement for fractionation and hence reduce overall costs of treatment, which to a large extent is proportional to the number of treatment fractions per patient.
A particularly important role for radioprotectors has emerged from the recognition that accelerated repopulation of tumour cells during radiotherapy can seriously compromise the effectiveness of treatment. The main consequences of this have been as follows:                (i) The development of accelerated treatment schedules to reduce the overall time of radiotherapy treatment. In such accelerated schedules, acute reactions are a particular problem. For example, acute oral mucositis in head and neck cancer patients indicates a clear need for radioprotectors.        (ii) The recognition that the interruption of radiotherapy treatment due to normal tissue reactions will reduce the probability of tumour control. Accordingly, the use of radioprotectors to prevent toxicity-induced treatment interruption would be clearly beneficial.        
The events of 11 Sep. 2001 prompted assessments of vulnerability to many types of terrorism scenarios, amongst which is a collection described as radiological terrorism. An example is the so-called “dirty bomb” involving dispersal of some form a radioactivity with conventional explosive. Whilst attention is focused on the acute radiation syndrome (ARS; also referred to as “radiation sickness”), which describes the consequences of whole-body exposure to radiation doses greater than 1 Gy, there are also concerns about the longer-term effects of low doses, namely radiation-induced mutagenesis and carcinogenesis (1). This general situation, and the realisation that no prophylactic agents are available to provide protection against exposure to ionising radiation, has generated significant research and political activity.
The mean lethal dose of radiation required to kill 50% of humans 60 days after whole-body irradiation (LD50/60) is between 3.25 and 4 Gy without supportive care, and 6-7 Gy when antibiotics and transfusion support are provided (1). The mortality is largely attributed to the haematopoietic syndrome, a consequence of hypoplasia or aplasia of the bone marrow. Cytopenias develop as a result of radiation-induced and normal attrition of mature functional cells, combined with the failure of replacement because of radiation-induced depletion of haematopoietic stem cells and progenitors. The time and extent of cytopenia generally correlate with radiation dose and prognosis, but the kinetics of depletion and recovery of blood cells also varies between the erythropoiesis, myelopoiesis and thrombopoiesis lineages, thrombopoiesis being the slowest.
The gastrointestinal syndrome results from ablation of stem cells in intestinal crypts, which in turn leads to denudation of the intestinal mucosa. This injury occurs after whole-body doses in the range of 3-15 Gy and in rodents doses at the upper end of this range usually result in death within about 1 week after irradiation.
Countermeasures against unplanned irradiation include a wide range of potential molecular and cellular interventions. However, the mechanistic simplicity of chemical radioprotection—that is, reduction of radiation-induced DNA damage—is attractive because of its widespread potential. In this context, the possible need for protection of individuals at risk of exposure to low radiation doses, to thereby minimise long-term radiation effects such as mutagenesis and carcinogenesis, is particularly important. Such individuals would include emergency personnel involved in response to unplanned exposures, as well as those subject to occupational exposure to ionising radiation.
A further group would be patients exposed to ionizing radiation during diagnostic medical procedures conducted in diagnostic radiology and nuclear medicine departments of hospitals and outpatient facilities.
The radioprotective properties of the minor groove binding DNA ligand Hoechst 33342 were first described by Smith, P. J. and Anderson, C. O. (2), who used clonogenic survival assays of irradiated cultured cells. Young, S. D. and Hill, R. P. (3) reported similar effects in cultured cells, but extended their studies to in vivo experiments. They concluded that the lack of radioprotection in their in vivo experiments was due to insufficient levels of Hoechst 33342 being delivered to target cells following intravenous injection. The findings of Hill and Young underline an important requirement for effective radioprotectors, namely potency. If the radioprotector is more potent, then it is more likely to achieve the required concentrations in an in vivo setting.
There is another aspect to be considered apart from potency. The concentration required for radioprotection must be non-toxic regardless of the potency of the radioprotector. If the radioprotector is delivered systemically, then this lack of toxicity requirement includes not just the cells and tissues to be protected from the radiation, but extends to the toxicity to the subject as a whole. In the case of Hoechst 33342 toxicity limits the extent to which it is useful as a radioprotector.
There is also a substantial conceptual problem in using radioprotectors in cancer radiotherapy. In attempting to decrease the effect of radiation on normal tissues by application of radioprotectors, there is a fear that some of the radioprotector will reach the tumour, thereby compromising tumour cell kill. The existing radioprotectors, e.g. WR2721 (Amifostine) and its active metabolite WR1065, are relatively small, diffusible molecules which do not avidly bind to tissue components and can therefore penetrate effectively through cell layers, so that they can reach the tumour via the circulation.
There is a need for radioprotectors that have limited penetration through cell layers. Such a property enables radioprotectors to be applied locally or topically to critical radiosensitive normal tissues in the vicinity of the tumour. Limited penetration restricts the extent to which the radioprotector reaches the capillary bed and is taken up into the circulation thereby reaching the tumour by systemic delivery in sufficient concentrations to confer significant radioprotection to the tumour.
The limited diffusion of DNA-binding ligands such as Hoechst 33342 through cell layers is known and has been exploited in mapping the location of cells in multi-cellular spheroids and in vivo, with respect to perfusion. Thus perfusion of Hoechst 33342 is considered a surrogate marker for perfusion of oxygen. In addition to restricting access to the tumour by systemic uptake following local or topical application to normal tissues, there is a further potential advantage of limited penetration in the context of cancer radiotherapy. This advantage stems from the view that the vasculature, in particular the endothelial cells, are the critical targets that determine the damaging effects of radiation. Furthermore, most radioresistant cells in the tumour are those viable cells that are most distant from the capillaries. The radioresistance of these cells is due to their hypoxic state, which in turn reflects their remoteness from the capillaries.
Consequently, radioprotectors having limited diffusion, when administered intravenously, will be delivered more efficiently to critical radiosensitive cells in normal tissues, than to the hypoxic subpopulation of cells in tumours which limit the effectiveness of radiotherapy generally. Thus, the use of such radioprotectors would be expected to enable higher radiation doses to be used, with increased probability of killing the hypoxic cells in the tumour.
However, the potential of the combination of these radiobiological features and the characteristics of DNA-binding radioprotectors can only be useful in cancer radiotherapy provided that an over-riding and necessary requirement of the radioprotectors exists, namely that the radioprotectors are sufficiently potent as to confer demonstrable radioprotection at non-toxic concentrations, when applied topically or systemically. A further practical requirement is that the extent of the limited penetration is sufficient to prevent significant systemic uptake following topical application, but not so pronounced so as to prevent sufficient concentrations from reaching the cells that determine the radiosensitivity of the tissue to be protected from the effects of ionising radiation, by topical or local application.
The extent of radioprotection (in the contexts of both cancer radiotherapy and protection from unplanned radiation exposure) is generally described in terms of dose modification factor (DMF), which is defined as the ratio of radiation doses required to produce the equivalent radiation-induced effect (molecular, cellular or in vivo endpoint) in the presence and absence of the radioprotector. When the radioprotective effect is observed on the basis of an in vivo endpoint, mechanisms other than modification of the initial radiation-induced damage may be involved. For example, for both the haematopoietic syndrome and the gastrointestinal syndrome, infection plays an important role in ultimate mortality, as a consequence of neutropenia and breach of the intestinal mucosal barrier, respectively. Thus, some immunostimulants have potential as mitigators of the radiation response. Immunostimulants can also be effective post-irradiation.
International patent publication No. WO97/04776 and the subsequent publication by Martin et at (4) disclose certain bibenzimidazole compounds characterised by substitution with sterically hindering and electron donating groups. Although these compounds demonstrate strong radioprotective activity there is scope to reduce the inherent cytotoxicity of compounds of this general class. The challenge, however, is to do so while retaining, and preferably improving, radioprotective activity (measured as dose modification factor). The disclosures of WO97/04776 are included herein in their entirety by way of reference.
International patent publication No. WO/2008/074091 also discloses bibenzimidazole compounds substituted with fluorine and/or chlorine and which, relative to known radioprotector compounds such as those of International patent publication No. WO97/04776, exhibit reduced cytotoxicity activity. While the cytotoxicity of the fluorine and chlorine substituted bibenzimidazole compounds was improved there is still a need for development of alternative radioprotective compounds, and preferably compounds that can be used in cancer radiotherapy, in protection of biological material from effects of radiation exposure and/or in protection of humans or animals from the effects of unplanned irradiation, which demonstrate low cytotoxicity but that retain radioprotective potency, and preferably that penetrate through cell layers to a limited extent. In particular it is desirable in some contexts that such compounds can be administered topically to protect tissues such as the skin, oral mucosa, oesophageal mucosa, rectal mucosa, vaginal mucosa and bladder epithelium, as well as parenterally to protect organs such as the lung and brain.